The eradication of global pathogens, including malaria, hinges upon the development of effective vaccines. One of the key strategies to realize successful vaccines is to identify and develop a new mechanism-based adjuvant. ?-galactocylceramide (?-GalCer), a glycolipid that has been extensively investigated, is known to display a significant biological activity, including an adjuvant effect, by binding CD1d molecules and stimulating invariant NKT (iNKT) cells, an important sub-population of the immune response. Recently, we identified a novel ?-GalCer analog, 7DW8-5, which can strongly enhance the level of malaria-specific protective immune response upon its intra-muscular (i.m.) co-administration with a candidate malaria vaccine. When compared to ?-GalCer, its parental glycolipid, we found the adjuvant effect of 7DW8-5 to be clearly superior. 7DW8-5 exhibited not only a stronger stimulatory activity towards iNKT cells and dendritic cells (DCs), but also a higher binding affinity to both mouse and human CD1d molecules than ?-GalCer. Very surprisingly, we discovered that when administered by i.m. injection, only ?-GalCer, but not 7DW8-5, induced a systemic production of cytokines including IFN-? and IL-12, whereas both glycolipids induced a similar level of systemic cytokine production upon their intravenous administration. We hypothesize that 7DW8-5 is retained locally at the i.m. injection site and nearby draining lymph nodes due its higher binding affinity to CD1d molecules and/or the up- regulation of CD1d expression level on activated DCs by 7DW8-5. In Aim 1, therefore, we will first determine whether 7DW8-5 is indeed retained locally and if so, how 7DW8-5 is prevented from spreading out and inducing systemic cytokine production. We also suspect that its local retention is directly related to the superior adjuvant effect exerted by 7DW8-5 and hypothesize that an increased activation of DCs induced by 7DW8-5 results in a stronger onset of the adaptive immune response elicited by vaccines. We will test this hypothesis in Aim 2. Finally, it is still unclear whether 7DW8-5 could indeed enhance humoral and CD4+ T cell responses and whether these responses could contribute to the protection against malaria. Therefore, using mice lacking CD8+ T cells and/or antibodies, we will determine these issues in Aim 3. In view of our recent studies showing that 7DW8-5 provides a significant adjuvant effect on the cellular immunogenicity of an adenovirus-based malaria vaccine in non-human primates, and because 7DW8-5 is advancing into a Phase 1 clinical trial in 2012, the proposed studies should be imperative for the future clinical applications of 7DW8-5 not only as an adjuvant for various vaccines, but also as a direct therapeutic agent.